Cancer treatment with retroviral vectors comprising wild-type p53

Abstract

Disclosed are methods and compositions for the selective manipulation of gene expression through the preparation of retroviral expression vectors for expressing antisense sequences, such as K-ras oncogene antisense sequences, or sequences encoding a desired product, such as wild type p53 sequences. Preferred retroviral vectors of the present invention incorporate the β-actin promoter in a reverse orientation with respect to retroviral transcription. Preferred antisense RNA constructs of the present invention employ the use of antisense intron DNA corresponding to distinct intron regions of the gene whose expression is targeted for down-regulation. In an exemplary embodiment, a human lung cancer cell line (NCI-H460a) with a homozygous spontaneous K-ras mutation was transfected with a recombinant plasmid that synthesizes a genomic segment of K-ras in antisense orientation. Translation of the mutated K-ras mRNA was specifically inhibited, whereas expression of H-ras and N-ras was unchanged. A three-fold growth inhibition occurred in H460a cells when expression of the mutated ras p21 protein was down-regulated by antisense RNA and cells remained viable. The growth of H460a tumors in nu/nu mice was substantially reduced by expressed K-ras antisense RNA.

1. A method for treating cancer in a human patient comprising directly introducing into a p53-deficient tumor cell of the patient a retroviral composition comprising a retroviral vector construct in a pharmaceutically acceptable buffer, wherein said retroviral vector construct comprises a wild-type p53 gene coding region, the coding region being operatively linked to a promoter to effect expression of the coding region, wherein said expression is effective to inhibit the growth of said tumor cell. 
     
     
       2. The method of  claim 1 , wherein the human patient has an epithelial cancer. 
     
     
       3. The method of  claim 2 , wherein the human patient has lung cancer. 
     
     
       4. The method of  claim 3 , wherein the patient has non-small cell lung cancer. 
     
     
       5. The method of  claim 1 , wherein the human patient is administered both a wild-type p53 gene coding region and an antisense K-ras gene coding region. 
     
     
       6. The method of  claim 1 , wherein the promoter and gene coding region are positioned in an orientation that is opposite that of retroviral transcription. 
     
     
       7. The method of  claim 1 , wherein the vector comprises a marker gene. 
     
     
       8. The method of  claim 4 , wherein the non-small cell lung cancer is sqamous cell cancer. 
     
     
       9. The method of  claim 4 , wherein the non-small cell lung cancer is adenocarcinoma. 
     
     
       10. The method of  claim 4 , wherein the non-small cell lung cancer is large-cell undifferentiated. 
     
     
       11. The method of  claim 3 , wherein the lung cancer is small cell lung cancer. 
     
     
       12. The method of  claim 1 , wherein the promoter is selected from the group consisting of β-actin, CMV, RSV, N2A, LN, LNSX, LNSN, LNCX and SV40. 
     
     
       13. The method of  claim 12 , wherein the promoter is β-actin. 
     
     
       14. The method of  claim 12 , wherein the promoter is CMV. 
     
     
       15. The method of  claim 1 , wherein said introducing is via intratumoral injection. 
     
     
       16. The method of  claim 1 , wherein said introducting is via circumferential injection of said tumor. 
     
     
       17. The method of  claim 1 , further comprising tumor resection. 
     
     
       18. The method of  claim 17 , wherein said resection is via bronchoscopy. 
     
     
       19. The method of  claim 17 , wherein said introducing is via injection of a resected tumor site. 
     
     
       20. The method of  claim 16 , wherein said injection is submucosal. 
     
     
       21. The method of  claim 16 , wherein said injection is subcutaneous. 
     
     
       22. The method of  claim 1 , wherein said introducing is regional. 
     
     
       23. The method of  claim 1 , wherein said introducing is performed multiple times. 
     
     
       24. The method of  claim 23 , wherein said introducing is performed daily for five consecutive days. 
     
     
       25. The method of  claim 23 , wherein said introducing is performed monthly. 
     
     
       26. The method of  claim 1 , further comprising photographing said tumor mass prior to introducing said retroviral composition. 
     
     
       27. The method of  claim 1 , wherein said retroviral composition is delivered in 10 ml. 
     
     
       28. The method of  claim 1 , wherein said retroviral composition is delivered in 0.1 ml. 
     
     
       29. The method of  claim 1 , wherein said retroviral composition has a titer of at least 10 5  CFU/ml. 
     
     
       30. The method of  claim 29 , wherein said retroviral composition has a titer of at least 10 6  CFU/ml. 
     
     
       31. The method of  claim 30 , wherein said retroviral composition has a titer of at least 6×10 6  CFU/ml. 
     
     
       32. The method of  claim 31 , wherein said retroviral composition has a titer of at least 9×10 6  CFU/ml. 
     
     
       33. The method of  claim 1 , wherein said retroviral vector is derived from MMTV or MMLV. 
     
     
       34. The method of  claim 1 , wherein said tumor mass is endobronchial. 
     
     
       35. The method of  claim 1 , wherein said retroviral composition further comprises protamine. 
     
     
       36. The method of  claim 1 , wherein said protamine is present at a concentration of 5 μg/ml. 
     
     
       37. The method of  claim 1 , further comprising removing tumor cells from said patient, introducing said retroviral composition ex vivo and returning retrovirally-treated cells to said patient. 
     
     
       38. The method of  claim 37 , wherein said tumor cells are bone marrow-derived.